Abstract Neuroblastoma is a complex and clinically challenging pediatric malignancy. For advanced disease, autologous hematopoietic stem cell transplantation (HSCT) has resulted in significantly better event-free survival as compared to maintenance chemotherapy alone. However, even with the use of tandem autologous HSC transplants, overall survival rates are still typically less than 50%. New therapeutic approaches are needed. Our preclinical data indicates that a multi-faceted approach using autologous HSCT as a platform for immunotherapy can be used to eliminate established neuroblastoma. This approach includes adoptive T cell transfer (cellular immunotherapy) and tumor vaccination. Data generated in our laboratory has indicated that anti-tumor efficacy can be further optimized through graft manipulation or through blockade of immune suppressive mechanisms. We found that when T cells added to the HSC graft were pre-sensitized to tumor antigens, in vivo-depletion of CD4 T cells increased anti-tumor efficacy, resulting in the complete elimination of established tumors. However, while early anti-tumor efficacy was increased in the absence of CD4 cells, generation of CD8 T cell memory was severely compromised. We hypothesize that early anti-tumor immunity can be enhanced after pre-sensitized T cell transfer through ex vivo depletion of CD4 T cells, or CD4 subsets, without the loss of CD8 memory. Our laboratory has also recently identified an interesting gene signature, upregulation of stem cell-associated genes in the CD8 cell compartment, which occurs very early after induction of 'productive' anti-tumor immunity. We hypothesize that this upregulation of stemness genes is an early signature for developing T cell memory. We speculate that the stemness gene signature in T cells may also serve as an early predictor of effective immunity. To test our hypotheses we propose two specific aims. In the first aim we will more closely examine the cellular requirements and vaccine schedule required for achieving effective and long-lasting anti-tumor immunity. This will involve an examination of the CD4 cell compartment to determine if immune suppressive CD4 cells can be selectively eliminated to achieve enhanced anti-tumor immunity without loss of CD8 memory. As an alternative approach, we will explore strategies to restore CD8 memory once tumors have been eliminated following CD4 depletion. In the second aim, we will examine the biological relevance of vaccine-induced T cell stemness, and identify the T cells responsible for this gene signature. Our goals are to learn how to optimize this multi-faceted approach to immunotherapy and to understand the mechanisms associated with successful treatment so that it can be used to reproducibly eliminate established disease. The ultimate goal of these preclinical studies is the development of effective immunotherapy for patients with neuroblastoma.